C O M M U N I C A T I O N S
in proximity to Arg38 and His33. In contrast, t-BuOOH was found to
occupy a different site on cyt c in proximity to His18. These results
imply that FFA-OOHs undergo a heterolytic reaction as opposed to
the homolytic reaction mechanism exhibited by organic hydroperox-
ides, further corroborating the results obtained experimentally.
Overall, the reaction of cyt c with hydroperoxides may proceed
via both homo- and heterolytic cleavage of an O-O bond. However,
the contribution of the heterolytic mechanism to the reduction of
FFA-OOH is overwhelmingly greater than that of the homolytic
pathway. Simultaneous involvement of two- and one-electron
-OOH reductions is not unique for cyt c/CL complexes; peroxidase
catalysis by cyclooxygenase-2 utilizes both pathways in a 60:40
ratio.18 Utilization of FFA-OOHs by cyt c/CL complexes in vivo
may be an interesting new pathway in the synthesis of oxygenated
fatty acids. Our previous studies identified these complexes and
oxidation of polyunsaturated species of CL as a required step in
execution of the apoptotic program.4 The source(s) of oxidizing
equivalents feeding the peroxidase cycle, however, have not been
identified. We further established that cyt c also catalyzes phos-
pholipase A-like reaction to hydrolyze peroxidized CLs and release
oxygenated FFAs.19 The current results identify the FFA-OOHs
as potent sources of oxidizing equivalents that maintain high levels
of peroxidase activity and production of sufficient amounts of
oxidatively modified CLs. Physiological role(s) of hydroxy deriva-
tives of FFA warrant further studies.
Figure 2. Spin trapping of 13(S)HpODE-, 4HpNE- and t-BuOOH-derived
radicals generated by cyt c/CL under conditions of equal consumption of
oxidizing equivalents when cyt c was preincubated with (A) CL/PC
liposomes and DMPO, (B) PC liposomes and DMPO, and (C) CL/PC
liposomes and POBN.
induced the formation of DMPO adducts. In contrast, t-BuOOH
produced strong EPR signals when CL/PC or PC liposomes were
incubated with cyt c. DMPO/ ·OC(CH3)3 radical adduct (aN ) 14.90
G, aHꢀ ) 16.04 G, and aHγ ) 0.63 G) was detected with PC
liposomes. The major signal detected in the presence of cyt c/CL
complexes was represented by the decomposition product of DMPO
adducts [DMPOX, aN ) 7.25 G, aHꢀ (2H) ) 4.05 G].
Further, hydroperoxide-derived radicals were analyzed using another
spin trap, POBN, which has been successfully utilized for the detection
of lipid-centered radicals.16 Again, a strong EPR signal was detected when
cyt c/CL complexes were incubated with t-BuOOH (10 mM) (Figure 2C).
The signal was represented by a sum of signals, the major of which had
splitting constants of aN ) 15.7 G and aH ) 2.7 G characteristic of adducts
with carbon-centered radicals.16 No discernible signals were observed with
either 4HpNE or 13(S)HpODE. The results of the spin-trapping experi-
ments suggest that cyt c/CL peroxidase complexes split FFA-OOHs (in
contrast to t-BuOOH) predominantly via the heterolytic mechanism.
The major products of heterolytic reduction of hydroperoxides
are hydroxy compounds, while a broad spectrum of oxygenated
decomposition products may be formed via the free-radical-
mediated homolytic pathway. Electrospray ionization mass spec-
trometry (ESI-MS) analysis demonstrated that 13-hydroxy-(9Z,11E)-
octadecadienoic acid (13-HODE) and other possible products of
the heterolytic pathway (m/z 295, 311, and 327; Figure 3) were
the major products generated by cyt c/CL from 13(S)HpODE (65
( 3% of total products), thus confirming the predominance of the
heterolytic reaction mechanism.
Acknowledgment. This work was supported by grants from the
NIH (U19-AI068021, HL70755, R03TW007320, 2RO1LM007994-
05), the NSF (CC0449117), the PittGrid, and la Junta de Extremadura,
Orden 2008050288 (A.K.S.A.).
Supporting Information Available: Experimental details, calcula-
tions of peroxidase reaction rate constants, prediction of t-BuOOH,
4HpNE, and 13(S)-HpODE binding sites, and complete ref 4. This
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Figure 3. Homo- and heterolytic enzymatic conversion of 13(S)HpODE
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